Genetics Ch 7,8,9,+10

Restriction enzymes (RE)

Cut DNA into fragments so they can be ligated into a plasmid (AKA Restriction Endonucleases)

Vector

Artifically constructed DNA molecule that can replicate in a host organism

Recombinant DNA

Foreign DNA +vector

EcoRI, BamHI, PStI

RE's

Occurrence of Restriction sites formula

(1/4)^n

Ligase function

joins DNA fragments and forms phosphodiester bonds

Ori

Origen of replication in plasmid vector

Selectable Marker

Gene that gives cell a selectable phenotype (ex. Ampicillin resistance gene)

MCS

Multiple cloning sites, contains unique RE sites where foreign dna is inserted. Ex. LacZ gene

CDNA library

Complimentery DNA libraries only represent protein coding regions. Much smaller and easier to work with

Dideoxy sequencing

based on DNA pol extending a short primer (oligo) using cloned DNA as a template

Oligo

Primer used for dideoxy sequencing. Can be radioactive for tracking. Anneals to template

ddNTP's

Dideoxynucleotides, lack 3'-OH and are unable to bond with 5'-phosphates of other nucleotides (end the chain)

PCR

Polymerase Chain Reaction, produces many copies of a specific DNA sequence, used to amplify DNA for cloning, used in forensics

Taq polymerase

Thermys aquaticus: a polymerase from thermophilic bacterium that makes PCR possible

three steps to PCR (with temps)

1. Dentature 95° 2. Anneal 50° 3. Extend 72°

PCR is useful for:

amplifing DNA for cloning, amplifing a mutant gene, amplifing human gene with mouse primers, amplifing dna for genomic sequencing

How much can PCR amplifty?

up to 40 Kb

ORF

Open reading Frame: Encodes about 50-100 aa's

EST

expressed sequence tag: good for identifying introns. One shot (about 500 bp's) sequencing of DNA library clone

BLAST

Basic local alignment search tool: online programs that looks for homogoly between query and database sequences

Reverse genetics

Obtain mutant via Knockout (replaced with resistant marker). Ethical limitations. (CRISPR is a knockout method)

transscriptomics

whole genome study of gene expresssion (when/what/how much a gene is expressed)

Micro array

each ORF fixed on a spot

Proteomics

global study of protein expression, modifications, protein-protein interaction, and function

When searching for a clone in a DNA library:

Plate # then well #, often there is little info unless it is a model organism that is well sequenced

cDNA Library uses

Screening expressed sequences, look for gene active in specific type of tissue, when you have raised an antibody against that protein

to screen in a cDNA library a ___ is needed

expression vector. Use an Antibody to find your intended protein from expression vector

Expression vector

vector that allows for txn and tln of a cloned gene, contains promotor and terminator for functional gene expression.

cDNA library is made from ___

Recombinant DNA vectors using transformed bacteria and microtiter plates

cDNA library copied cells are lysed ___ (where)

"in situ": in their original places with their proteins bound to the filter

Radioactively Labeled Antibody

An antibody that binds to the protein of interest during incubation of cDNA libraries. Form dark spots that can be found with an X-Ray

What a genomic clone has that a cDNA clone doesn't

Promoters and introns (cDNA is only expressed genes)

difference between screening in a genomic DNA library vs a cDNA library:

filter is incubated with labeled ssDNA which form hybrids with complementary ssDNA molecules bound to the filter. Detects DNA with similar radioactive ssDNA probe

Heterologous probes

identify DNA in libraries. Probe is made of DNA from another organism that closely matches function of target DNA (more closely related, better accuracy)

NaOH

lyses cells for cDNA library copies

Oligonucleotide

Synthetic ssDNA used as a probe to reverse engineer DNA sequence from protein sequence (about 20 nt's each)

Guessmer

mixture of oligonucleotides

Complementation of mutations

when a mutant gene is 'copied over' by a functional (wild type) gene. Vectors are selected for via media supplementation.

Shuttel vector

capable of replicating in 2 or more organisms. Used in complementation of mutations.

Restriction mapping

determines order and distance of RE sites in a segment of DNA

Agarose gel

used to visualize DNA strand lengths in restriction mapping

EtBr

used to stain DNA for restriction mapping, floresces under UV light (can cause indel mutations)

Marker fragments

gel electrophoreses ladder. Used for comparison of samples

Restriction mapping is useful for:

Subcloning and confirming results (ensuring proper orientation)

Subcloning

Moving a segment of a clone to another vector (isolate target genes)

Southern Blot (1975)

DNA fragments are transferred from gel to a filter and hybridized with a probe. determines copy number in genome, compares homologous genes, screens individuals with diseases associated with RE cutting pattern.

NaOH (southern blot)

denatures DNA to ssDNA

Souther blot can and can't use

Can use vector, PCR, etc. Can't use whole genomic library

Northern Blot

Uses uncut mRNA, determines size of mRNA coded by gene from different tissues (location on gel), gene activity (thickness in gel)

Which of the following stumualtes the transpostion of the Ty element in Gerry Fink's experiment?

a. Glucose

b. galactose

c. sucrose

d. an intron

B. Galactose

Most common RE's recognize palindrones of ___(# of bp's)

4,6, or 8-bp

On an agar containing ampicillin and X-gal, a lac-Z- bacterial host that has uptaken 'pBSII+insert" should be:

a. blue

b. white

c. no colonies

b. white

Ori is required for

a. DNA synthesis

b. RNA synthesis

c. protein synthesis

d. antibiotic selection

e. DNA insertion

A. DNA synthesis Ori= Origen of DNA replication

WHich of the following techniques involves partial digestion?

a. Genomic library construction

b. cDNA library construcuton

A. Genomic library construction

cDNA came directly from

a. genomic DNA

b. mRNA

c. could be either

b. mRNA reverse transcriptase uses mRNA to make DNA

Content of a genomic library microtiter plate is made up of frozen

a. cDNA

b. RNA

c. bacteria

d. proteins

c. bacteria (bacteria hosts contain vector + insert)

The extension cycles of PCR take place at

a. 95 C

b. 72 C

c. 60 C

d. 37 C

b. 72 C

Sanger sequencing is possible because ddNTP lacks

a. 2'H

b. 2'OH

c. 3'H

d. 3'OH

e. 5' phosphate

D. 3' OH

"In vitro" means

a. inside an organism

b. in a test tube

c. in place

d. inside a cell

e. within living

B. in a test tube ex. PCR is done in vitro

Which of the Following requires an expression vector?

a. Genomic Library Screening

b. the antibody screening approach

c. the guessmer approach

d. two of the above

e. all of the above

b. the antibody screening approach

The guessmer approach is appropriate when you have

a. a partial peptide sequence

b. a gene from a related species

c. an expression vector

d. a mutant

e. none of the above is useful

a. partial peptide sequence -guess DNA sequence

___ is a protein used by the immune system to neutralize foreign substances

a. antigen

b. antibody

c. probe

b. antibody

What year was the DNA double helix model proposed?

1953

Northern blot can quantify the amount of proteins produced in a cell type. T/F?

b. False (RNA's)

Transposable elements (TE's)

DNA segments that can move from one location to another

Transposition

movements of TE's, involves non-homologous recombination with insertion into DNA that has no sequence homology with the TE's

(In prokaryotes) Where can TE move to? (3 answers)

Another position on same chromosome, a plasmid, a phage genome

Insertion sequences (IS)

simplest TE found in prokaryotes

Transposase

gene for mobilization and insertion of DNA. Recognizes IR and cuts the IS off from donor DNA and Cuts target site DNA

Inverted repeats (IR)

identical (or near-identical) sequence at the terminals, in opposite orientations (PRO)

Host enzymes

(DNA polymerase and ligase) fill the gaps left by sticky ends

Size range of Prokaryotic IS

768bps to over 5kb

Transposons (Tn)

similar to IS, but carry additional genes (in addition to transposase), and are more complex, produce target site duplications (makes staggered cut at host DNA) (PRO)

Composite transposons

Carry genes (e.g. antibiotic resistance) flanked on both sides by IS elements

ISL and ISR

Insertion sequence left and right may be inverted or direct orientation

Noncomposite trasposons

also carry genes but do NOT terminate with IS elements

Resolvase

promotes a recombination event associated with transposition

Conservative transposition

the Tn is lost from its original position when it transposes

Replicative transposition

The Tn is duplicated (both donor and recipient have 1 copy)

Class II (DNA elements)

moves directly from donor site to target site, can excise from donor site and reverse the mutation.

Class I (retroelements)

-an RNA intermediate is made from this element via txn

-DNA is made from the RNA via reverse txn

-DNA is then inserted into target site

-remain permanently once inserted into the target

-the copy # of class I element can be large, since many RNAs can be transcribed from a single element

Ty elements or retrotransposon (class I)

Ty encodes two proteins and two repeats

Gag

a structural protein that packages the RNA intermediate into a virus-like particle

Pol

responsible for reverse txn and integration

Long terminal repeats (LTR)

each contains a promoter and sequences recognized by pol

Retrotransposition

transposition via an RNA intermediate

inducible promotor

promoter that can be controlled by scientists. Used in Fink's experiment, galactose activated the promotor he used

Ac-Ds elements

(Class II) Activator and dissociation elements discovered in 1951.

Ac (activator)

4563 bp long with short IR and a transposase. Autonomous

Ds (Dissociation)

generated by Ac by deletions. contain IR's but notransposase, mutated versions of Ac, nonautonomous, require Ac for mobilization

LINEs and SINEs

human retrotransposons (Class I), do not have LTRs, caused by insertional mutagenesis. (EU)

Difference between Class I and II elements

Class II are conservative and Class I are replicative and have RNA intermediates

Difference between composite and non composite transposons

Composite transposons are flanked on both sides by IS elements while Non composite transposons do not terminate with an IS element (they end in an IR, and require more genes to be able to jump, and are replicative)

Pro vs Eukaryotic transposition differences

Prokaryotes have IS elements and transposons while Eukaryotes have DNA elements and retrotransposons

Both Prokaryotes and Eukaryotes make what kinds of cuts?

Staggered so that the two ends and left 'sticky'

Which of the following is a (genetic definition) palindrome?

A. GATTAG

B. GAATTC

C. AATTCG

B. GAATTC Genetic palindromes when copied and read in the same direction result in the original sequence

Reverse Genetics

Starts with the gene and works out its function. Ethical limitations